Botulism is caused by botulinum neurotoxin secreted by members of the genus Clostridium and is characterized by flaccid paralysis, which if not immediately fatal, requires prolonged hospitalization in an intensive care unit and mechanical ventilation. Naturally occurring botulism is found in infants or adults whose gastrointestinal tracts become colonized by Clostridial bacteria (infant or intestinal botulism), after ingestion of contaminated food products (food botulism), or in anaerobic wound infections (wound botulism) (Center for Disease Control (1998) Botulism in the United States, 1899-1998. Handbook for epidemiologists, clinicians, and laboratory workers. Atlanta, Ga. U.S. Department of Health and Human Services, Public Health Service: downloadable at www.bt.cdc.gov/agent/botulism/index.asp). Botulism neurotoxins (BoNTs) are also classified by the Centers for Disease Control (CDC) as one of the six highest-risk threat agents for bioterrorism (the “Category A agents”), due to their extreme potency and lethality, ease of production and transport, and need for prolonged intensive care (Arnon et al. (2001) JAMA 285: 1059-1070). Both Iraq and the former Soviet Union produced BoNT for use as weapons (United Nations Security Council (1995) Tenth report of the executive committee of the special commission established by the secretary-general pursuant to paragraph 9(b)(I) of security council resolution 687 (1991), and paragraph 3 of resolution 699 (1991) on the activities of the Special Commission; Bozheyeva et al. (1999) Former Soviet biological weapons facilities in Kazakhstan: past, present, and future. Center for Nonproliferation Studies, Monterey Institute of International Studies), and the Japanese cult Aum Shinrikyo attempted to use BoNT for bioterrorism (Arnon et al. (2001) supra). As a result of these threats, specific pharmaceutical agents are needed for prevention and treatment of intoxication.
No therapies are available for prevention or treatment of botulism, and only an investigational pentavalent toxoid vaccine is available from the CDC (Siegel (1988) J. Clin. Microbiol. 26: 2351-2356) and a recombinant vaccine is under development (Smith (1998) Toxicon 36: 1539-1548). Regardless, mass civilian or military vaccination is unlikely due to the rarity of disease or exposure and the fact that vaccination would prevent subsequent medicinal use of BoNT. Post-exposure vaccination is useless, due to the rapid onset of disease. Toxin neutralizing antibody (Ab) can be used for pre- or post-exposure prophylaxis or for treatment (Franz et al. (1993) pp. 473-476 In B. R. DasGupta (ed.), Botulinum and Tetanus Neurotoxins: Neurotransmission and Biomedical Aspects. Plenum Press, New York). Small quantities of both equine antitoxin and human botulinum immune globulin exist and are currently used to treat adult (Black and Gunn. (1980)/ii. J. Med., 69: 567-570; Hibbs et al. (1996) Clin. Infect. Dis., 23: 337-340) and infant botulism (Arnon (1993) Clinical trial of human botulism immune globulin., pp. 477-482. In B. R. DasGupta (ed.), Botulinum and Tetanus Neurotoxins: Neurotransmission and Biomedical Aspects. Plenum Press, New York) respectively.
Recombinant monoclonal antibody (mAb) could provide an unlimited supply of antitoxin free of infectious disease risk and not requiring human donors for plasmapheresis. Given the extreme lethality of the BoNTs, mAbs must be of high potency in order to provide an adequate number of doses at reasonable cost. The development of such mAbs has become a high priority research aim of the National Institute of Allergy and Infectious Diseases. While to date no single highly potent mAbs have been described, it has been reported that combining two to three mAbs could yield highly potent BoNT neutralization (Nowakowski et al. (2002) Proc. Natl. Acad. Sci. USA, 99: 11346-50).
The development of mAb therapy for botulism is complicated by the fact that there are at least seven BoNT serotypes (A-G) (Hatheway (1995) Curr. Top. Microbio. Immunol, 195: 55-75.) that show little, if any, antibody cross-reactivity. While only four of the BoNT serotypes routinely cause human disease (A, B, E, and F), there has been one reported case of infant botulism caused by BoNT/C (Oguma et al. (1990) Lancet 336: 1449-1450), one outbreak of foodborne botulism linked to BoNT/D (Demarchi, et al. (1958) Bull. Acad. Nat. Med., 142: 580-582), and several cases of suspicious deaths where BoNT/G was isolated (Sonnabend et al. (1981) J. Infect. Dis., 143: 22-27). Aerosolized BoNT/C, D, and G have also been shown to produce botulism in primates by the inhalation route (Middlebrook and Franz (1997) Botulinum Toxins, chapter 33. In F. R. Sidell, E. T. Takafuji, D. R. Franz (eds.), Medical Aspects of Chemical and Biological Warfare. TMM publications, Washington, D.C), and would most likely also affect humans. Thus it is likely that any one of the seven BoNT serotypes can be used as a biothreat agent.
Variability of the BoNT gene and protein sequence within serotypes has also been reported and there is evidence that such variability can affect the binding of monoclonal antibodies to BoNT/A (Kozaki et al. (1998) Infect. Immun. 66: 481 1-4816; Kozaki et al. (1995) Microbiol. Immunol. 39: 767-774).
Antibodies for treatment or prevention of botulism must be able to protect against the major forms of botulinum toxin (A, B, and E) (Simpson (1996) Annas Internal Med. 125 (7):616-7). They also must have high potency to provide an adequate number of doses at reasonable cost. Together with the anti-BoNT/A antibodies, antibodies to BoNT/B and BoNT/E could provide protection against all of the major forms of botulism.
Although a large number of antibodies binding different epitopes on BoNTs have been examined, potent toxin neutralization by a single antibody has not been observed. As demonstrated for BoNT/A toxin, extremely potent neutralization of BoNTs B and E toxins can be achieved by combining antibodies to generate mixtures, typically of three antibodies, which act in a synergistic manner to potently neutralize these toxins. A putative requirement for antibody synergy to occur is that the antibodies bind different toxin epitopes so that multiple antibodies can be attached to BoNT leading to rapid Fc-mediated systemic clearance. Multiple antibodies have a higher probability of being bound to BoNT when the affinity of each individual antibody for BoNT is high.
Thus, the prevention and treatment of botulism may particularly benefit from the development of a formulation containing multiple antibodies. However, for the reasons described below, stable formulations containing multiple antibodies have yet to be realized.
Because proteins are larger and more complex than traditional organic and inorganic drugs (i.e. possess multiple functional groups in addition to complex three-dimensional structures), the formulation of such proteins can be problematic. For a protein to remain biologically active, a formulation must preserve intact the conformational integrity of at least a core sequence of the protein's amino acids while at the same time protecting the protein's multiple functional groups from degradation. Degradation pathways for proteins can involve chemical instability (i.e. any process which involves modification of the protein by bond formation or cleavage resulting in a new chemical entity) or physical instability (i.e. changes in the higher order structure of the protein). Chemical instability may be caused by deamidation, racemization, hydrolysis, oxidation, beta elimination or disulfide exchange. Physical instability may be caused by denaturation, aggregation, precipitation or adsorption, for example. The three most common protein degradation pathways are protein aggregation, deamidation and oxidation (Cleland et al. (1993) Critical Reviews in Therapeutic Drug Carrier Systems 10 (4):307-377).
Antibody molecules, as part of the group of protein pharmaceuticals, are very susceptible to physical and chemical degradation, such as denaturation and aggregation, deamidation, oxidation and hydrolysis. Protein stability is influenced by the characteristics of the protein itself, e.g. the amino acid sequence, and by external influences, such as temperature, solvent pH, excipients, interfaces, or shear rates. It is thus important to define the optimal formulation conditions to protect the protein against degradation reactions during manufacturing, storage and administration (Manning et al. (1989) Pharm. Res. 6 (11): 903-18).
Most pharmaceutical antibody compositions may comprise single monoclonal antibodies such as HERCEPTIN®, HUMIRA® etc. In some instances, administration of multiple monoclonal antibodies directed to a single target or multiple targets and administrated in combination may improve their diagnostic or therapeutic indication and efficacy. For example, in a collagen-induced arthritis model, an anti-TNFα antibody was shown to be more effective in combination with either an anti-IL-1R antibody or an anti-CD4 antibody (Williams et al. (2000) J. Immunol. 2000, 165:7240-45). Cocktails of three or more antibodies binding simultaneously to a cytokine e.g. IL-6, interferon-α have been proposed as a means of enhancing clearance of a target molecule overcoming the problem of accumulation of monomeric immune complexes (Montero-Julian et al. (1995) Blood 85 (4):917-24; Kontsek et al. (1991) Immunol. 73:8-11). For some indications, such as infectious diseases, multiple antibodies that target different epitopes on a single target or different targets (for example, different toxins or infectious agent or different subtypes of the same toxin) may be necessary to achieve therapeutic efficacy. Oligoclonal cocktails comprising multiple monoclonal antibodies have been described (reviewed in Logtenberg (2007) Trends in Biotechnol. 25 (9):390-94) but are generally limited by their short-term stability. For example U.S. Pat. No. 6,262,790 describes a cocktail of two monoclonal antibodies directed to different conserved epitopes on prion proteins reportedly having broad reactivity to PrP proteins in spite of interspecies and intraspecies variation. Other examples include two—antibody cocktails for rhesus D or Idiopathic Thrombocytopenic Purpura (ITP) (U.S. Pat. No. 5,851,524; symphogen.com), rabies (Bakker et al. (2005) J. Virol. 79 (14):9062-68; de Kruif et al. (2007) Annu Rev. Med. 58:359-68; International Patent Publ. Nos. WO 05/118644 and WO 08/068,246), hepatitis C (Eren et al. (2006) J. Virol. 80: 2654-64), hepatitis B (International Patent Publ. No. WO 06/112838), Shiga toxin (International Patent Publ. No. WO 07/143,004), EGF-R-positive cancers (International Patent Publ. No. WO 08/104,183) and breast cancer (Nahta et al. (2004) Cancer Res. 64: 2343-46); three-antibody cocktails for HIV (Xu et al. (2002) Vaccine 20: 1956-60) and botulinum neurotoxin type A (BoNT/A) (Nowakowski et al. (2002) Proc. Natl. Acad. Sci. USA 99 (17): 11346-50; International Patent Publ. No. WO 2005/016232); and a five-antibody cocktail for rabies (European Patent Publ. No. EP 0 402 029). Approaches for producing recombinant polyclonal antibodies have also been described (Haurum & Bregenholt (2005) IDrugs 8 (5):404-409, Rasmussen et al. (2007) Biotechnol. Lett. 29: 845-52; International Patent Publ. No. WO 06/007853). International Patent Publ. No. WO 98/01476 describes mixtures of polyclonal and monoclonal anti-HIV antibodies.
The stabilization of polypeptides in pharmaceutical compositions remains an area in which trial and error plays a major role (reviewed by Wang (1999) Int. J. Pharm. 185:129-88). Numerous factors can be varied in order to find suitable excipients and optimal conditions for preparing a long-term stable formulation for a single monoclonal antibody, making this a challenging process. Stably formulating two different antibodies in a single formulation is even more problematic and involves choosing excipients and conditions that represent a compromise. These difficulties are compounded for formulating three antibodies, or more.
The development of a stable formulation for multiple antibodies also requires determination of stability and degradation of the individual antibodies present in the antibody mixture. Such a determination is often difficult due to the large number of antibodies in the formulation and their similarities.
This invention addresses and overcomes these difficulties and provides related advantages as well.